Method of preparing pertussis toxin and toxoid



Patented Oct. 3, 1944 by METHOD OF PREPARING rna'russrs 'roxm AND 'roxom Merritt E. Roberts and Edwin F. Void, Pearl River, N. Y., assignors to Lederle Laboratories, Inc., New York, N. 1

a corporation of Dela- No Drawing. Application February 13, 1941, I Serial No. 378,772

I '7 Claims.

This invention relates to pertussis toxin and toxdid and to methods of preparing the same.

The study of whooping cough from a scientific as well .as clinical standpoint has been carried out by many workers with generally indiflerent success. The production of a relatively stable antigen suitable for use in immunization against whooping'cough'and for the treatment thereof has been the subject of extensive research. The need for such materials is apparent in view of the prevalency of this common ailment. The production of such a material capable of stimulating measurable antibodies has been especially. de-

sirable.

We have discovered a method of preparing pertussis toxin and toxoid which products have been found effective in their ability to stimulate the production of titrable antibodies in the human being. The production of toxin is accomplished by growing suitable cultures of Bacteriaceae Hemophilus pertussis under properly controlled conditions and in suitable media from which a potent toxin can be obtained. This potent toxin can be suitably detoxifled with preservation of antigen to give the corresponding toxoid.

The following specific example'is given merely by way of illustration and not in limitation:

- A suitable culture medium for the production of potent toxin is prepared by adding to a beef heart infusion base, about 2% peptone, about 0.05% gastric mucin, 0.1% soluble starch (e. g. potato starch) 0.5% sodium chloride and 0.05% disodium phosphate. The pH of this solution is adjusted to 7.8 and the medium is then placed in suitable containers. Thus we may use fiveliter lots in nine-liter bottles, giving a culture medium between about 5" and 9" in depth. The culture medium is sterilized by placing the contamer and medium in a suitable autoclave or the like and heating for a suitable period of time at a sterilizing temperature.

to be used as such, it is best stored at low temperatures. The toxin can be left for months without deterioration in'the frozen and dried state; subjecting the toxin to a temperature of 50 C. for nine minutes-destroys its potency and, at higher temperatures, destruction takes place in shorter periods of time. These properties as well as tests made on rabbits and mice indicate that this product is apparently a true antigenic toxin and not an endotoxin, a non-antigenic toxic substance.

In order to prepare a toxoid of maximum antigenicity from the toxin just described, we add formalin in a suitable amount dependent upon 30 C. for four to seven days.

the strength of the toxin. As illustrative, about .3% (by volume) formalin (40% formaldehyde infection by the, further addition of suitable pre- Two-day old seed culture of H. pertussis is washed off a blood agar slant and inoculated into the sterilized medium in the bottles. The air in the. bottles is displaced by a mixture of about 20% carbon dioxide and'80% oxygen and a similar mixture is passed through the culture medium during the growing period which last from four to ten days at about 32-3'7C. At that time the contents of therbottle are removed and subjected to centrifugationto separate the growth from the toxin solution. The clear solution so obtained is filtered in a Berkefeld filter.

.servative materials if necessary.

The pertussis toxin prepared as described above has been found to be neutralized by the antibodies in serum from children who have re-! toxoid may be used for immunization against whooping cough or for the treatment thereof. This toxoid stimulates the production of antibodies ln the blood of a child so that his serum will neutralize pertussis toxin.

The atmosphere in which the incubation and growth takes place. is preferably one containing a. mixture of carbon dioxide and oxygen in suitable quantities although the proportions thereof are not necessarily limited to those given in-the specific example. However,-the growth may take place in the presence of ordinary air, although toxins of lower potency are generally obtained, when compared with cultures maintained for the same periods of time in the atmosphere containing carbon doxide.

The time and temperature for incubation are also subject to some variation although the times stated are considered the optimum. While longer periods of time may be utilized for the incubation, such extended periods may cause undesirable side reactions with the formation of autolytic products which are considered undesirable.

The filtrate constitutes the toxin and when it is Our toxin and toxoid have proved themselves covered from whooping cough. The pertussis maximum in about seventy-two hours.

em active in tests on rabbits and human beings.

When our toxin is injected intradermally into rabbits, there is produced a characteristic reaction at the site of injection which reaches a The undiluted toxin causes necrosis somewhat similar to that produced by the injection of whole cultures. The toxoid produced by our detoxification treatment, u'pon injection into rabbits, produces antibodies which neutralize pertussis toxin, as evidenced by injections of such toxin. Our toxoid stimulates production of neutralizing antibodies for the necrotic factor of whole cultures as evidenced by the lack of necrosis when live cultures are injected into rabbits previously immunized with toxoid. It is evidentthat our toxold injections stimulate the production of a true antitoxin.

The composition of the culture medium used for the production of the pertussis toxin need not be restricted to all of the specific materials and proportions stated in the example. In place of the beef heart infusion, any other suitable meat infusion may be used such as beef infusion, horse infusion and the like, or .this' may be omitted entirely. The nature of the nutrient medium may account for variability in yield of toxin, it being found that the beef heart infusion isgenerally preferred since it gives constant and high yields of toxin. The soluble starch and peptone are important ingredients although the exact nature and degree of purity of these materials may be varied somewhat. The chloride and phosphate assist somewhat in the production of. the toxinbut may be varied in amount or, if desired, may even be omitted entirely. The preferred composition of the culture medium should be such that production of pertussis toxin and toxoid and these products have been'proved clinically to be effective. The present process is an improvement on the process described in that application and permits the preparation of more concentrated toxins and consequently stronger toxoids. As already noted, the toxin solutions prepared in accordance with the present process average about 50 units M. L. D. without concentration. The

.Voigt and Phillips process results in a toxin containing about 10 to 20 units M. L. D. It will be obvious that the more potent toxin has many advantages and is more desirable than the less potent material. This is especially true when the toxin is converted to the corresponding toxoid and this toxoid is utilized in the formation of a pertussis antitoxin. However, this antitoxin is more particularly described and claimed in copending application of Roberts-and Ospeck, Serial No. 381,664 filed March 4, 1941, v

The pertussis toxin made can be standardized by using this aforementioned antitoxin. Standardization is carried out by determining the amount of toxin which is neutralized by a designated amount of a standard, stable antitoxin. Neutralization is determined by injection of titrated mixtures of different amounts of toxin with a fixed amount of antitoxin into the skin of susceptible animals (e. g. rabbits) to find the point at which ther is no specific toxin lesion. The mixture which gives this effect indicates the neutralization value of the toxin. This test is considered quite accurate since the antitoxin is a stable standard and since the skin test is effective with relatively small amounts of toxin. Micecan be used for the same purpose provided enough excess toxin is added to the neutralization mixture to insure one or more M. L. D.s of unneutralized toxin. The rabbit intradermal test will detect about .025 to .05 M. L. D. of unneutralized toxin.

The toxoids of the present invention have been found to be valuable antigens-for the immunizaexpressed as units M. L. D. (minimum lethal dose). One M. L. D. of pertussis toxin is that amount of toxin which, when administered intravenously in white mice of 16 to 18 grams weight,

will. cause death within four days in at least one half of the total number of mice injected. Usually six mice are used for each test. The toxin prepared in accordance with the present procedure averages about units M. L. D. or, in other words, 1 part of our toxin may be diluted to /50 of its strength and the diluted solution will be sufllciently potent to 'kill at least three mice out of six injected intravenously with this solution in accordance with the test just de-, scribed. It is to be understood, 'of course, that the amount injected in each case is exactly the Serial No. 159,912, filed August 19, 1937, now

Patent No. 2,240,969 dated May 8, 1941, there is described and claimed a related method for the tion against whooping cough or for treatment of the disease. The amount of toxoid injected varies in accordance with the relative potency of the toxoid. The antibody response in children,

' casional and negligible rise in body temperature.

As illustrative of the dosage, a toxoid prepared from our toxin containing units M. L. D., may be administered in two injections, 1 co. in each arm. The injection is preferably repeated two or three more times at one week intervals, making a total of three or four injections in each arm. After such treatment it is found that the normal child has 6 to 10 protection units per cc. of blood and this is believed to be significant in view of the fact that normal children have 0.4 or less protection units per cc. of blood and children convalescing from the disease pertussis have from 3 to 5 protection units per cc. of blood.

Other suitable changes and variations may be made in carrying out our invention without departing from the spirit and scope thereof as defined in the appended claims.

We claim:

1. The process which comprises growing a culture of pertussis bacilli in a deep culture medium of about pH 7.4-7.8 containing small amounts of soluble starch and peptone, passing a stream of gas containing carbon dioxide through the meg assasss dium while maintaining the medium at a temass containing carbon dioxide through the me-.

perature of about 32-37 C. for about four to.

ten days until maximum formation of pertussis toxin is obtained without the formation of substantial amounts of autolytic products, separating the toxin solution from the medium and detoxifying the toxin by adding a small amount of formaldehyde thereto and heating at about 40 C. for about one week.

4. The process which comprises growing a culture of pertussis bacilli in a deep culture medium of about pH 7.4-7.8 containingmeat infusion together with small amounts of soluble starch and peptone, passing a stream.of gas containing asubstantial amount of carbon dioxide through the medium while maintaining the medium at a temperature 0! about 8247 C. for about four to ten days until maximum formation of pertussis toxin is obtained without the formation of substantial amounts of autolytio products.

ture of pertussis bacilli in a deep culture medium of about pH 7.4-7 .8 containing meat infusion together with small amounts of soluble starch and peptone, passing a stream of gas containing carbon dioxide through the medium while maintaining the medium at a temperature oi about 32-37 C. for about four to ten days until maximum formation of pertussis toxin is obtained without the formation of substantial amounts of autolytic products, separating the toxin'solution .from the medium and detoxifying the toxin by adding a small amount of formaldehyde thereto and heating at about 40 C. for about one week.

6. The process which comprises growing a culture of pertussis bacilli in a deep culture medium of about pH 7.4-7.8 containing meat infusion togather with small amounts of soluble starch and peptone, passing a stream of gas containing carbon dioxide through the medium while maintaining the medium at a temperature of about 32-37 C. for about four to ten days until maximum formation of pertussis toxin is obtained without the formation of substantial amounts of autolytic products, separating the toxin solution from the medium and detoxifying the toxin.

7. The process which comprises growing a culture oi pertussis bacilli in a deep culture medium of about pH 7.4-7.8 containing beef heart infusion together with small amounts of soluble starch and peptone, passing a stream of gas containing carbon dioxide through the medium while maintaining the medium at a temperature of about 32-37 C. for about four to ten days until maximum formation of pertussis toxin is obtained without the formation of. substantial amounts or autolytic products, separating the toxin solution from the medium and detoxifying the toxin by adding a small amount of formaldehyde thereto and heating at about 40' ,C. for

40 about one week.

5. The process which comprises growing a cult 

